Characterization of renin mRNA expression and enzyme activity in rat and mouse mesangial cells

Renin is an enzyme involved in the stepwise generation of angiotensin II. Juxtaglomerular cells are the main source of plasma renin, but renin activity has been detected in other cell types. In the present study we evaluated the presence of renin mRNA in adult male Wistar rat and mouse (C-57 Black/6) mesangial cells (MC) and their ability to process, store and release both the active and inactive forms of the enzyme. Active renin and total renin content obtained after trypsin treatment were estimated by angiotensinogen consumption analyzed by SDS-PAGE electrophoresis and quantified by angiotensin I generation by HPLC. Renin mRNA, detected by RT-PCR, was present in both rat and mouse MC under basal conditions. Active renin was significantly higher (P<0.05) in the cell lysate (43.5 ± 5.7 ng h-1 10(6) cells) than in the culture medium (12.5 ± 2.5 ng h-1 10(6) cells). Inactive prorenin content was similar for the intra- and extracellular compartments (9.7 ± 3.1 and 3.9 ± 0.9 ng h-1 10(6) cells). Free active renin was the predominant form found in both cell compartments. These results indicate that MC in culture are able to synthesize and translate renin mRNA probably as inactive prorenin which is mostly processed to active renin inside the cell. MC secrete both forms of the enzyme but at a lower level compared with intracellular content, suggesting that the main role of renin synthesized by MC may be the intracellular generation of angiotensin II.

Chronic converting enzyme inhibition normalizes QT interval in aging rats

The aim of the present study was to investigate the effects of converting enzyme inhibition by captopril on ECG parameters in aged rats. Four-month-old male rats received captopril dissolved in tap water (0.5 mg/l) or tap water for 2 or 20 months. At the end of treatment, under anesthesia, RR and PR interval, P wave and QRS duration, QT and corrected QT interval were measured in all animals. On the following day, chronic ECG (lead II) recordings were performed to quantify supraventricular (SVPB) or ventricular premature beats (VPB). After sacrifice, the hearts were removed and weighed. RR interval was similar in young and untreated aged rats, but significantly larger in aged rats treated with captopril. P wave and QRS length did not differ among groups. PR interval was significantly larger in old than in young rats and was not affected by captopril. Corrected QT interval was larger in aged than in young rats (117 ± 4 vs 64 ± 6 ms, P<0.05) and was reduced by captopril (71 ± 6 ms, P<0.05). VPB were absent in young rats and highly frequent in untreated old animals (8.4 ± 3.0/30 min). Captopril significantly reduced VPB in old rats (0.3 ± 0.1/30 min, P<0.05). The cardiac hypertrophy found in untreated aged rats was prevented by captopril (3.44 ± 0.14 vs 3.07 ± 0.10 mg/g, P<0.05). The beneficial effects of angiotensin converting enzyme inhibition on the rat heart during the aging process are remarkable.

Insulin secretion, insulin sensitivity, and hepatic insulin extraction in first-degree relatives of type 2 diabetic patients

To identify early metabolic abnormalities in type 2 diabetes mellitus, we measured insulin secretion, sensitivity to insulin, and hepatic insulin extraction in 48 healthy normal glucose-tolerant Brazilians, first-degree relatives of type 2 diabetic patients (FH+). Each individual was matched for sex, age, weight, and body fat distribution with a person without history of type 2 diabetes (FH-). Both groups were submitted to a hyperglycemic clamp procedure (180 mg/dl). Insulin release was evaluated in its two phases. The first was calculated as the sum of plasma insulin at 2.5, 5.0, 7.5, and 10.0 min after the beginning of glucose infusion, and the second as the mean plasma insulin level in the third hour of the clamp procedure. Insulin sensitivity index (ISI) was the mean glucose infusion rate in the third hour of the clamp experiment divided by the mean plasma insulin concentration during the same period of time. Hepatic insulin extraction was determined under fasting conditions and in the third hour of the clamp procedure as the ratio between C-peptide and plasma insulin levels. FH+ individuals did not differ from FH- individuals in terms of the following parameters [median (range)]: a) first-phase insulin secretion, 174 (116-221) vs 207 (108-277) µU/ml, b) second-phase insulin secretion, 64 (41-86) vs 53 (37-83) µU/ml, and c) ISI, 14.8 (9.0-20.8) vs 16.8 (9.0-27.0) mg kg-1 min-1/µU ml-1. Hepatic insulin extraction in FH+ subjects was similar to that of FH- ones at basal conditions (median, 0.27 vs 0.27 ng/µU) and during glucose infusion (0.15 vs 0.15 ng/µU). Normal glucose-tolerant Brazilian FH+ individuals well-matched with FH- ones did not show defects of insulin secretion, insulin sensitivity, or hepatic insulin extraction as tested by hyperglycemic clamp procedures.

Dipeptidyl peptidase IV (CD26) activity in the hematopoietic system: differences between the membrane-anchored and the released enzyme activity

Dipeptidyl peptidase IV (DPP-IV; CD26) (EC 3.4.14.5) is a membrane-anchored ectoenzyme with N-terminal exopeptidase activity that preferentially cleaves X-Pro-dipeptides. It can also be spontaneously released to act in the extracellular environment or associated with the extracellular matrix. Many hematopoietic cytokines and chemokines contain DPP-IV-susceptible N-terminal sequences. We monitored DPP-IV expression and activity in murine bone marrow and liver stroma cells which sustain hematopoiesis, myeloid precursors, skin fibroblasts, and myoblasts. RT-PCR analysis showed that all these cells produced mRNA for DPP-IV. Partially purified protein reacted with a commercial antibody to CD26. The K M values for Gly-Pro-p-nitroanilide ranged from 0.43 to 0.98 mM for the membrane-associated enzyme of connective tissue stromas, and from 6.76 to 8.86 mM for the enzyme released from the membrane, corresponding to a ten-fold difference, but only a two-fold difference in K M was found in myoblasts. K M of the released soluble enzyme decreased in the presence of glycosaminoglycans, nonsulfated polysaccharide polymers (0.8-10 µg/ml) or simple sugars (320-350 µg/ml). Purified membrane lipid rafts contained nearly 3/4 of the total cell enzyme activity, whose K M was three-fold decreased as compared to the total cell membrane pool, indicating that, in the hematopoietic environment, DPP-IV activity is essentially located in the lipid rafts. This is compatible with membrane-associated events and direct cell-cell interactions, whilst the long-range activity depending upon soluble enzyme is less probable in view of the low affinity of this form.

Nitric oxide regulates angiotensin-I converting enzyme under static conditions but not under shear stress

Mechanical forces including pressure and shear stress play an important role in vascular homeostasis via the control of the production and release of a variety of vasoactive factors. An increase in vascular shear stress is accompanied by nitric oxide (NO) release and NO synthase activation. Previously, we have demonstrated that shear stress induces angiotensin-I converting enzyme (ACE) down-regulation in vivo and in vitro. In the present study, we determined whether NO participates in the shear stress-induced ACE suppression response. Rabbit aortic endothelial cells were evaluated using the NO synthase inhibitor L-NAME, and two NO donors, diethylamine NONOate (DEA/NO) and sodium nitroprusside (SNP). Under static conditions, incubation of endothelial cells with 1 mM L-NAME for 18 h increased ACE activity by 27% (from 1.000 ± 0.090 to 1.272 ± 0.182) while DEA/NO and SNP (0.1, 0.5 and 1 mM) caused no change in ACE activity. Interestingly, ACE activity was down-regulated similarly in the presence or absence of L-NAME (delta(0 mM) = 0.26 ± 0.055, delta(0.1 mM) = 0.21 ± 0.22, delta(1 mM) = 0.36 ± 0.13) upon 18 h shear stress activation (from static to 15 dyn/cm²). Taken together, these results indicate that NO can participate in the maintenance of basal ACE levels in the static condition but NO is not associated with the shear stress-induced inactivation of ACE.

Antioxidant status of dog aqueous humor after extracapsular lens extraction

We determined the antioxidant status of the aqueous humor after extracapsular lens extraction in 14 mongrel dogs weighing about 10 kg. The animals were examined by slit lamp biomicroscopy, applanation tonometry and indirect ophthalmoscopy. One eye was submitted to conventional extracapsular lens extraction and the other was used as control. Samples of aqueous humor were obtained by anterior chamber paracentesis before and at days 1, 2, 3, 7 and 15 after surgery. Total antioxidant status was determined as the capacity of aqueous humor to inhibit free radical generation by 2,2-azobis(2-amidopropane) chlorine. Ascorbic acid concentration was measured by HPLC with UV detection. Protein content was determined with the biuret reagent. Statistical analysis was performed by ANOVA followed by the Tukey-Kramer test. Protein concentration increased from 0.61 to 22 mg/ml 24 h after surgery. These levels were maintained and returned to normal at day 7. Total antioxidant capacity was reduced from 50 to about 30 min until day 3 and at day 7 it was equal to control. Ascorbic acid levels were reduced from 252 to about 110 µM and then returned to control values at day 15. Considering the importance of ascorbic acid concentration in aqueous humor for the maintenance of the antioxidant status of the anterior segment of the eye, the decrease of antioxidant defenses suggests that the surgical procedures promote an oxidative stress condition in the eye.

DNA extraction and quantification from touch and scrape preparations obtained from autopsy liver cells

The objective of the present study was to develop a simplified low cost method for the collection and fixation of pediatric autopsy cells and to determine the quantitative and qualitative adequacy of extracted DNA. Touch and scrape preparations of pediatric liver cells were obtained from 15 cadavers at autopsy and fixed in 95% ethanol or 3:1 methanol:acetic acid. Material prepared by each fixation procedure was submitted to DNA extraction with the Wizard® genomic DNA purification kit for DNA quantification and five of the preparations were amplified by multiplex PCR (azoospermia factor genes). The amount of DNA extracted varied from 20 to 8,640 µg, with significant differences between fixation methods. Scrape preparation fixed in 95% ethanol provided larger amount of extracted DNA. However, the mean for all groups was higher than the quantity needed for PCR (50 ng) or Southern blot (500 ng). There were no qualitative differences among the different material and fixatives. The same results were also obtained for glass slides stored at room temperature for 6, 12, 18 and 24 months. We conclude that touch and scrape preparations fixed in 95% ethanol are a good source of DNA and present fewer limitations than cell culture, tissue paraffin embedding or freezing that require sterile material, culture medium, laboratory equipment and trained technicians. In addition, they are more practical and less labor intensive and can be obtained and stored for a long time at low cost.

In situ enzyme immunoassay for titration of a Brazilian hepatitis A virus strain (HAF-203)

Hepatitis A virus (HAV) replicates relatively slowly in cell culture without a cytopathic effect, a fact that limits the use of tissue culture assays. The radioimmunofocus assay is the standard method for HAV titration, although it is labor intensive and requires the use of radioisotopes. A simple, rapid and objective infectivity assay based on an in situ enzyme immunoassay (EIA) is described here for a Brazilian cell culture-adapted HAV strain (HAF-203). The assay uses a peroxidase-labeled polyclonal antibody to fixed monolayers as an indicator of infection. EIA may be completed within 7 days using serial 5-fold dilutions of the virus, yielding a titer of 5.024 log 50% tissue culture infective dose (TCID50)/ml for HAF-203. This technique had a detection limit of 1.1 log TCID50/ml and the specificity was demonstrated by detecting no reaction on the columns of uninfected wells. The reproducibility (with intra- and inter-assay coefficients of variation ranging from 1.9 to 3.8% and from 3.5 to 9.9%, respectively) and quantitation of the assay were demonstrated by close agreement in virus infectivity titers among different assays of the same amount of virus and between assays of different amounts of virus. Furthermore, this assay does not require the use of radiolabeled antibodies. We describe here an efficient EIA that is highly reproducible and that could be used to monitor HAV growth in cell culture and to determine the quantity of HAV antigen needed for diagnostic assays. This is the first report of the infectious titer of the Brazilian cell culture-adapted HAV strain (HAF-203).

Control of the rat angiotensin I converting enzyme gene by CRE-like sequences

We characterized the role of potential cAMP-responsive elements (CRE) in basal and in induced angiotensin converting enzyme (ACE) gene promoter activity in order to shed light on the regulation of somatic ACE expression. We identified stimulators and repressors of basal expression between 122 and 288 bp and between 415 and 1303 bp upstream from the transcription start site, respectively, using a rabbit endothelial cell (REC) line. These regions also contained elements associated with the response to 8BrcAMP. When screening for CRE motifs we found pCRE, a proximal sequence between 209 and 222 bp. dCRE, a distal tandem of two CRE-like sequences conserved between rats, mice and humans, was detected between 834 and 846 bp. Gel retardation analysis of nuclear extracts of REC indicated that pCRE and dCRE bind to the same protein complexes as bound by a canonical CRE. Mutation of pCRE and dCRE in REC established the former as a positive element and the latter as a negative element. In 293 cells, a renal cell line, pCRE and dCRE are negative regulators. Co-transfection of ATF-2 or ATF-2 plus c-Jun repressed ACE promoter activity, suggesting that the ACE gene is controlled by cellular stress. Although mapping of cAMP responsiveness was consistent with roles for pCRE and dCRE, mutation analysis indicated that they were not required for cAMP responsiveness. We conclude that the basal activity of the somatic ACE promoter is controlled by proximal and distal CREs that can act as enhancers or repressors depending on the cell context.

A continuous fluorescent assay for the determination of plasma and tissue angiotensin I-converting enzyme activity

A continuous assay using internally quenched fluorescent peptides with the general sequence Abz-peptidyl-(Dnp)P-OH (Abz = ortho-aminobenzoic acid; Dnp = 2,4-dinitrophenyl) was optimized for the measurement of angiotensin I-converting enzyme (ACE) in human plasma and rat tissues. Abz-FRK(Dnp)P-OH, which was cleaved at the Arg-Lys bond by ACE, was used for the enzyme evaluation in human plasma. Enzymatic activity was monitored by continuous recording of the fluorescence (lambdaex = 320 nm and lambdaem = 420 nm) at 37ºC, in 0.1 M Tris-HCl buffer, pH 7.0, with 50 mM NaCl and 10 µM ZnCl2. The assays can be performed directly in the cuvette of the fluorimeter and the hydrolysis followed for 5 to 10 min. ACE measurements in the plasma of 80 healthy patients with Hip-His-Leu and with Abz-FRK(Dnp)P-OH correlated closely (r = 0.90, P < 0.001). The specificity of the assay was demonstrated by the complete inhibition of hydrolysis by 0.5 µM lisinopril or captopril. Abz-FRK(Dnp)P-OH cleavage by ACE was monitored in rat lung, kidney, heart, and liver homogenates in the presence of a cocktail of inhibitors containing trans-epoxy-succinyl-L-leucylamido-(4-guanido)-butene, pepstatin, phenyl-methylsulfonyl fluoride, N-tosyl-L-phenylalanyl-chloromethyl ketone, and N-tosyl-lysyl-chloromethyl ketone to prevent undesirable hydrolysis. ACE activity in lung, heart and kidney homogenates, but not in liver homogenates, was completely abolished by 0.5 µM lisinopril or captopril. The advantages of the method are the procedural simplicity and the high sensitivity providing a rapid assay for ACE determinations.

Systemic and regional hemodynamic effects of enalaprilat infusion in experimental normotensive sepsis

Angiotensin-converting enzyme inhibitors have been shown to improve splanchnic perfusion in distinct shock states. We hypothesized that enalaprilat potentiates the benefits of early fluid resuscitation in severe experimental sepsis, particularly in the splanchnic region. Anesthetized and mechanically ventilated mongrel dogs received an intravenous infusion of live Escherichia coli over a period of 30 min. Thereafter, two interventions were performed: fluid infusion (normal saline, 32 mL/kg over 30 min) and enalaprilat infusion (0.02 mg kg-1 min-1 for 60 min) in randomized groups. The following groups were studied: controls (fluid infusion, N = 4), E1 (enalaprilat infusion followed by fluid infusion, N = 5) and E2 (fluid infusion followed by enalaprilat infusion, N = 5). All animals were observed for a 120 min after bacterial infusion. Mean arterial pressure, cardiac output (CO), portal vein blood flow (PVBF), systemic and regional oxygen-derived variables, and lactate levels were measured. Rapid and progressive reductions in CO and PVBF were induced by the infusion of live bacteria, while minor changes were observed in mean arterial pressure. Systemic and regional territories showed a significant increase in oxygen extraction and lactate levels. Widening venous-arterial and portal-arterial pCO2 gradients were also detected. Fluid replacement promoted transient benefits in CO and PVBF. Enalaprilat after fluid resuscitation did not affect systemic or regional hemodynamic variables. We conclude that in this model of normotensive sepsis inhibition of angiotensin-converting enzyme did not interfere with the course of systemic or regional hemodynamic and oxygen-derived variables.

Cytosine arabinoside-metabolizing enzyme genes are underexpressed in children with MLL gene-rearranged acute lymphoblastic leukemia

Infant acute lymphoblastic leukemia (IALL) is characterized by mixed lineage leukemia (MLL) gene rearrangements, unique gene expression profiles, poor prognosis, and drug resistance. One exception is cytosine arabinoside (Ara-C) to which IALL cells seem to be more sensitive. We quantified mRNA expression of Ara-C key enzymes in leukemic lymphoblasts from 64 Brazilian ALL children, 15 of them presenting MLL gene rearrangement, and correlated it with clinical and biological features. The diagnosis was based on morphological criteria and immunophenotyping using monoclonal antibodies. MLL gene rearrangements were detected by conventional cytogenetic analysis, RT-PCR and/or fluorescence in situ hybridization. The DCK and HENT1 expression levels were determined by real-time quantitative PCR using SYBR Green I. Relative quantification was made by the standard curve method. The results were analyzed by Mann-Whitney and Fisher exact tests. A P value of £0.05 was considered to be statistically significant. DCK and HENT1 expression levels were significantly lower in children with MLL gene-rearranged ALL compared to children with MLL germ line ALL (P = 0.0003 and 0.03, respectively). Our results differ from previous ones concerning HENT1 mRNA expression that observed a higher expression level in MLL gene-rearranged leukemias. In conclusion, the expression of the genes related to Ara-C metabolism was lower in MLL-positive children in the sample studied, suggesting the presence of population differences in the expression profile of these genes especially for HENT1.

Reduced plasma levels of angiotensin-(1-7) and renin activity in preeclamptic patients are associated with the angiotensin I- converting enzyme deletion/deletion genotype

The relationship between preeclampsia and the renin-angiotensin system (RAS) is poorly understood. Angiotensin I-converting enzyme (ACE) is a key RAS component and plays an important role in blood pressure homeostasis by generating angiotensin II (Ang II) and inactivating the vasodilator angiotensin-(1-7) (Ang-(1-7)). ACE (I/D) polymorphism is characterized by the insertion (I) or deletion (D) of a 287-bp fragment, leading to changes in ACE activity. In the present study, ACE (I/D) polymorphism was correlated with plasma Ang-(1-7) levels and several RAS components in both preeclamptic (N = 20) and normotensive pregnant women (N = 20). The percentage of the ACE DD genotype (60%) in the preeclamptic group was higher than that for the control group (35%); however, this percentage was not statistically significant (Fisher exact test = 2.86, d.f. = 2, P = 0.260). The highest plasma ACE activity was observed in the ACE DD preeclamptic women (58.1 ± 5.06 vs 27.6 ± 3.25 nmol Hip-His Leu-1 min-1 mL-1 in DD control patients; P = 0.0005). Plasma renin activity was markedly reduced in preeclampsia (0.81 ± 0.2 vs 3.43 ± 0.8 ng Ang I mL plasma-1 h-1 in DD normotensive patients; P = 0.0012). A reduced plasma level of Ang-(1-7) was also observed in preeclamptic women (15.6 ± 1.3 vs 22.7 ± 2.5 pg/mL in the DD control group; P = 0.0146). In contrast, plasma Ang II levels were unchanged in preeclamptic patients. The selective changes in the RAS described in the present study suggest that the ACE DD genotype may be used as a marker for susceptibility to preeclampsia.

Association of urinary 90 kDa angiotensin- converting enzyme with family history of hypertension and endothelial function in normotensive individuals

We described angiotensin-I-converting enzyme (ACE) isoforms with molecular masses of 190, 90, and 65 kDa in the urine of normotensive offspring of hypertensive subjects. Since they did not appear in equal amounts, we suggested that 90 kDa ACE might be a marker for hypertension. We evaluated the endothelial response in normotensive offspring with or without family history of hypertension and its association with the 90 kDa ACE in urine. Thirty-five normotensive subjects with a known family history of hypertension and 20 subjects without a family history of hypertension, matched for age, sex, body weight, and blood pressure, were included in the study. Endothelial function was assessed by ultrasound and a sample of urine was collected for determination of ACE isoforms. In the presence of a family history of hypertension and detection of 90 kDa ACE, we noted a maximal flow mediated dilation of 12.1 ± 5.0 vs 16.1 ± 6.0% in those without a previous history of hypertension and lacking urinary 90 kDa ACE (P < 0.05). In subjects with a family history of hypertension and presenting 90 kDa ACE, there were lower levels of HDL-cholesterol (P < 0.05) and higher levels of triglycerides (P < 0.05). Subjects with 90 kDa ACE irrespective of hypertensive history presented a trend for higher levels of triglycerides and HDL-cholesterol (P = 0.06) compared to subjects without 90 kDa ACE. Our data suggest that the 90 kDa ACE may be a marker for hypertension which may be related to the development of early atherosclerotic changes.